One important protein in primary hemostasis is von Willebrand Factor (VWF). Plasma von Willebrand Factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury, and especially, the very large VWF multimers are hemostatically competent. The existence of plasma factors that control the size of VWF multimers has long been suspected. The von Willebrand Factor-cleaving protease (“VWFCP”) is involved in the limitation of platelet thrombus growth by proteolytic cleavage of von Willebrand Factor multimers in humans (Furlan et al., Blood, 87:4223-4234 (1996)). Recently, the molecular structure of von Willebrand Factor-cleaving protease and the corresponding gene have been described (PCT App. Pub. No. WO 02/42441; Zheng et al., J Biol Chem, 276:41059-41063 (2001)) and have been identified as a new member of the ADAMTS family and designated ADAMTS13. VWFCP regulates VWF multimer size by proteolytic cleavage.
The large and ultra large VWF multimers play a central role in arterial thrombosis, whereby unusually large multimers of VWF have been seen in two similar forms of thrombotic microangiopathy—thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS)—both resulting in platelet aggregation leading to disseminated occlusions in the microcirculation. Patients with TTP have a deficiency of ADAMTS13, whereas patients with HUS show normal activity of the protease.
There are several types of TTP: an acute idiopathic or sporadic form, an intermittent form with an eventual relapse, and a chronic relapsing form. The rare hereditary form of TTP has been related to specific gene mutations in the ADAMTS13 locus. Acute idiopathic TTP or acquired TTP are usually more severe than chronic relapsing TTP, wherein these patients have acquired antibodies against ADAMTS13, which inhibit the von Willebrand Factor-cleaving protease (Furlan et al., Blood, 91:2839-2846 (1998); Furlan et al., N Engl J Med, 339:1578-1584 (1998)). TTP can be associated with autoimmune disorders, systemic infection, systemic malignancy, malignant hypertension, multiorgan failure, and congenital hemolytic anemia. TTP can initiate from events including: stem cell transplantation, pregnancy/postpartum, the administration of certain drugs, and bloody diarrhea. Intermittent relapsing TTP is also associated with the reappearance of ADAMTS13 inhibitor. For other forms of TTP, such as ticlopidine-associated TTP, it has also been observed that these patients have acquired antibodies against ADAMTS13 (Moake, N Eng J Med, 347:589-600 (2002)). However, some patients with acquired TTP having ultra large VWF multimers in plasma lack severely reduced levels of ADAMTS13.
Although TTP-like disorders have been associated with various medications, bone marrow transplantation, pregnancy, HIV infection, and autoimmune disease, most cases appear sporadically, without an obvious precipitating factor. This disease is seen most commonly in adults from 20 to 50 years old, with women affected slightly more often than men. In most TTP patients, the onset of the disease occurs in otherwise healthy individuals, and there is no history of a similar condition in other family members. However, in a smaller set of individuals, there is evidence suggesting that the condition can be inherited; and familial TTP has been reported in rare cases. In these cases, the disease begins early in life or sometime shortly after birth and is associated with multiple recurrences (i.e., a chronic relapsing course). The disease strikes about 4 out of every 100,000 people.
Current treatment consists of infusion of fresh frozen plasma with or without plasma exchange or plasmapheresis. In TTP, this treatment is repeated daily until blood tests show improvement. People who do not respond to this treatment, or who have frequent recurrences, can require removal of the spleen. Prior to the development of modern treatment protocols, fatality during an acute episode of TTP was greater than 90% (Rock et al., N Engl J Med, 325:393-397 (1991); George, Blood, 96:1223-1229 (2000)). Plasmapheresis has improved the outcome of this disease so that now 80 to 90% of patients recover completely; however, fatalities still occur. Although most incidents of the disease are acute, when relapses occur, the disease can become chronic.
Despite marked improvement in treatment outcome, the molecular pathogenesis of TTP is still unknown and the specific factor(s) responsible for the acute onset of this disease, or recovery following treatment, remains to be identified. Because the cause is unknown, there is no way to prevent the disease.
Existing diagnostic methods for ADAMTS13-related disorders such as TTP suffer from several limitations. For instance, most assays evaluate the activity of a subject's ADAMTS13 using substrates that are not endogenous to the subject. By failing to use the subject's own substrate, these methods fail to consider each of the relevant factors affecting the activity of ADAMTS13 for a particular subject. For instance, a subject's VWF can be resistance to cleavage by ADAMTS13, and this condition would go undetected by traditional diagnostic methods. Moreover, existing diagnostic methods generally require the application of shear stress or additional denaturing agents to induce ADAMTS13-mediated cleavage of VWF.
Therefore, a need exists in the art for improved ADAMTS13 activity assays, diagnostic methods for ADAMTS13-related disorders, and methods of evaluating the efficacy of treatment in individuals having ADAMTS13-related disorders. The present invention fulfills these needs.